There is widespread interest in efficient synthesis and screening of large numbers of compounds to identify candidate compounds with a given desired activity. [Ellman, J A and Gallop, M A, Curr Opin Chem Biol 2:317-319 (1998)].
Combinatorial libraries of random-sequence oligonucleotides, polypeptides, synthetic oligomers and small organic molecules have been described and their utility in identifying active compounds or as a starting point for developing related molecules with more desirable properties has been proposed (Ellman, J A and Gallop, M A, 1998).
One method for formation of combinatorial libraries involves preparation of high density position-addressable oligomer arrays on a planar substrate. In this method, a substrate having photoprotective groups is irradiated, using photolithographic mask techniques, in selected regions only, to deprotect surface active groups in those selected regions. The entire surface is then treated with a solution of a selected subunit, which itself has a photoprotected group, to react this subunit with the surface groups in the photodeprotected regions. This process is repeated to (i) add a selected subunit at each region of the surface, and (ii) build up different-sequence oligomers at known, addressable regions of the surface. [See, e.g., Fodor, S. P., et al., Science 251:767-773 (1991) and U.S. Pat. No. 5,143,854 (1992)].
This method has the advantage that reaction sites do not have to be physically separated during subunit addition, and therefore massive parallel subunit addition is possible by applying subunit-addition reagents over the entire surface of the array. Greater site density is therefore feasible than in systems where physical separation of reagents is required from one reaction site to another, and where individual reagents are spotted or deposited in defined array regions.
A related approach wherein the library is produced in capillary tubes has also been described wherein a method for producing, high-density, position-addressable combinatorial library of different-sequence oligomer or different-substituent small molecule compounds. The disclosed invention includes massive parallel synthesis of subunits and known, addressable library positions in a dense array of capillary tubes, and the screening of individual library compounds in either solution phase or solid phase. [U.S. Pat. Nos. 5,723,3204 (1998), 5,759,779 (1998), and 5,763,263 (1998)].
In a related approach, a traditional split-and-recombine strategy for synthesis of combinatorial libraries has been described. [Chen, et al., Methods in Enzymology 267:211-9 (1996); Ellman and Gallop, (1998)]. In one application of this approach, beads containing successive precursors to the target compounds that form the library may be alternately mixed and separated, with one of a selected number of reagents being added to each group of separated beads at each step [Furka, A., et al., Int. J. Pept. Protein Res. 37:487-493 (1991); Chen, C. et al., J. Am. Chem. Soc. 116:2661-2662 (1994); Pham, E. K. et al., PCT Intl. App. Pub. No. WO 9513538 (May/1995); Dillard, L. W. et al., PCT Intl. App. Pub. No. WO 9408051 (April/1994)]. An advantage of this method is that each bead contains only one chemical species, allowing the beads themselves to be used for screening. However, the identity of the species on each bead must be independently determined. Although several methods have been reported for tagging the support beads with molecules more readily analyzable than the library members themselves [e.g., Nestler, H. P. et al., J. Org. Chem. 59:4723-4724 (1994); Felder, E. et al., PCT Intl. Appn. Pubn. No. WO 9516209 (June/1995); Dillard, et al., 1994], the need for separate identification of each species nonetheless limits the usefulness of this approach for the preparation of very large libraries.
Replacements for the conventional bead support for combinatorial synthesis have also been described, e.g., use of linear homogeneous polymers such as polyethylene glycol chains [Janda and Han, Methods in Enzymol 267:234-247 (1996); Han et al. Proc. Nat Acad. Sci. USA 92(14):6419-6423 (1996)], and fluorinated hydrocarbon chains [Studer et al., Science 275(5301):823-826 (1997)]. On the basis of their solubility properties, these polymers have been exploited as selective “handles” to extract split-and-recombine library members from complex reaction mixtures. The various polymer supports useful in combinatorial library formation of same molecules have been recently reviewed. [Labadie, Curr Opin Chem Biol 2:346-352 (1998)].
Another general approach involves the synthesis of a combinatorial library as a physically segregated array of compounds [Geysen, H. M., et al., Proc. Natl. Acad. Sci. USA 81:39984002 (1984); Southern, E., EP Patent No. 373,203 (1994); Southern, E. et al., Genomics 13:1008-1017 (1992); Bunin, B. A., et al., J. Am. Chem. Soc. 114:10997-10998 (1992); Bunin, B. A., et al., Proc. Natl. Acad Sci. USA 91(11):4708 (1994); DeWitt, S. H. et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993)]. Libraries of compounds have been synthesized on functionalized resins either coated on (Geysen, et al, 1984, 1985; Bunin, et al., 1992, 1994) or contained within (DeWitt, et al., 1993) arrays of pins, with reactions carried out in separate chambers. Southern (1994) used arrays of spots laid down on a substrate such as glass by a pen plotter.
A key advantage of this approach is that the chemical identity of each library element on the array is associated with an addressable position on the array. However, in this method, as well as the split-mix method, preparation of very large libraries would require an inconvenient number of manipulations and/or a large array of separate reaction vessels or sites.
In cases where the compounds may be screened for biological activity while still attached to the substrate, this method also allows for massive and rapid screening, by binding a reporter-labeled target to the surface and determining the positions of bound target. Surface arrays of this type may be used both for combinatorial library screening (Fodor, S. P. A., et al., PCT Application WO 95/00530, published January, 1995; Geysen, et al., 1984, 1985) or for various types of oligonucleotide analysis, such as sequencing by hybridization (Drmanac, et al., 1993; Southern, 1994).
In a further approach, two alternating parallel combinatorial syntheses are performed such that a genetic tag is chemically linked to the chemical structure being synthesized. [See, e.g, Brenner and Lerner, Proc. Nat Acad. Sci. USA 89(12): 5381-5383 (1992); Lerner et al., U.S. Pat. No. 5,723,598 (1998)] In this method, the addition of a chemical unit is followed by the addition of an oligonucleotide sequence, which functions as an identifier for the structure of the chemical unit. A library is built up by the repeating the process after pooling and division of the reaction products obtained at each step.
One limitation in the early methods of combinatorial library formation is that large-library planar arrays are necessarily limited in the amount (number of molecules) of each library species, since the planar region available to each species is quite small, e.g., on the order of 102-103 μm2. As a consequence, the ability to detect binding species on the array may be limited. Further, it is not feasible to carry out solution-phase screening on a planar array, because of the difficulty of physically separating different array regions carrying different library members.
It would thus be desirable to provide a method for preparing a large combinatorial library of compounds which has the advantages of (i) massive parallel synthesis of subunits and known, addressable library positions, (ii) adaptable to virtually any oligomer or small-molecule chemistry, (iii) a relatively large area for synthesis of each library member, (iv) capable of being screened either as a mixture or as individual library compounds in either solution phase or solid phase, and (v) capable of amplifying and modifying selected library compounds.